Cloning of a Muscle-Specific Calpain from the American Lobster Homarus

Cloning of a muscle-speciﬁc calpain from the American lobster Homarus americanus: expression associated with muscle atrophy and restoration during moulting Xiaoli Yu and Donald L. Mykles* Department of Biology, Cell and Molecular Biology Program and Program in Molecular, Cellular and Integrative Neurobiology, Colorado State University, Fort Collins, CO 80523, USA *Author for correspondence (e-mail: [email protected])

Accepted 24 October 2002

Summary A cDNA (1977 bp) encoding a crustacean calpain (Ha- cord/thoracic ganglion and antennal gland. An antibody CalpM; GenBank accession no. AY124009) was isolated raised against a unique N-terminal sequence recognized a from a lobster fast muscle cDNA library. The open 62 kDa isoform in cutter claw and crusher claw closer reading frame specified a 575-amino acid (aa) polypeptide muscles and a 68 kDa isoform in deep abdominal muscle. with an estimated mass of 66.3 kDa. Ha-CalpM shared Ha-CalpM was distributed throughout the cytoplasm, as high identity with other calpains in the cysteine proteinase well as in some nuclei, of muscle fibers. Purification of Ha- domain (domain II; aa 111–396) and domain III (aa CalpM showed that the 62 kDa and 68 kDa isoforms co- 397–575), but most of the N-terminal domain (domain I; eluted from gel filtration and ion exchange columns at aa 1–110) was highly divergent. Domain II contained the positions consistent with those of previously described cysteine, histidine and asparagine triad essential for Ca2+-dependent proteinase III (CDP III; 59 kDa). Ha- catalysis, as well as two conserved aspartate residues that CalpM mRNA and protein did not change during the bind Ca2+. In domain III an acidic loop in the C2-like moulting cycle. The muscle-specific expression of Ha- region, which mediates Ca2+-dependent phospholipid CalpM and the ability of Ha-CalpM/CDP III to degrade binding, had an expanded stretch of 17 aspartate residues. myofibrillar proteins suggest that it is involved in Ha-CalpM was classified as a non-EF-hand calpain, as it restructuring and/or maintaining contractile structures in lacked domain IV, a calmodulin-like region containing five crustacean skeletal muscle. EF-hand motifs. Northern blot analysis, relative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that Ha-CalpM was highly Key words: calpain, calcium-dependent proteinase, muscle, atrophy, expressed in skeletal muscles, but at much lower levels in moulting, cDNA, gene expression, mRNA, lobster, Homarus heart, digestive gland, intestine, integument, gill, nerve americanus, Crustacea, Arthropoda.

Introduction Calpains constitute a large and diverse family of Ca2+- calmodulin-like domain containing five EF-hand motifs, the dependent cysteine proteinases in animal cells (Mykles, 1998; first three of which (EF-1, EF-2 and EF-3) bind Ca2+ with Sorimachi and Suzuki, 2001). In mammals, there are more than various affinities (Dutt et al., 2000; Hosfield et al., 1999). The a dozen calpain genes, some of which produce isoforms by N-terminal sequence (domain I) is the least conserved of the alternative mRNA splicing (Sorimachi and Suzuki, 2001). four domains; it varies in length and sequence between cDNAs encoding invertebrate calpains have been obtained different calpain genes (Sorimachi and Suzuki, 2001). At least from fruit fly (Emori and Saigo, 1994; Jékely and Friedrich, in some calpains, the N terminus acts as an inhibitory domain, 1999; Theopold et al., 1995), nematode (Sokol and Kuwabara, which is autocatalytically cleaved to activate the enzyme 2000), platyhelminth (Andresen et al., 1991) and trypanosome (Jékely and Friedrich, 1999). In addition to the Ca2+-binding (Hertz-Fowler et al., 2001). All calpains have a highly sites in domains III and IV, there are conserved aspartates in conserved catalytic domain (domain II), containing a cysteine, two non-EF-hand sites in domain II that bind Ca2+ and are histidine and asparagine that are essential for activity involved in full activation of the enzyme (Khorchid and Ikura, (Sorimachi and Suzuki, 2001). Domain III forms a C2-like 2002; Moldoveanu et al., 2002). structure with an acidic loop, which mediates Ca2+-dependent Lobster muscle contains four Ca2+-dependent cysteine phospholipid binding and activation (Hosfield et al., 1999, proteinase (CDPs I, IIa, IIb and III), or calpain, activities 2001; Sorimachi and Suzuki, 2001). Domain IV is a (Mykles and Skinner, 1986). All require Ca2+ at millimolar 562 X. Yu and D. L. Mykles levels for full activity in vitro and degrade myofibrillar protein Materials and methods (Mattson and Mykles, 1993; Mykles and Skinner, 1982, 1983, Cloning of cDNA encoding lobster muscle calpain (Ha- 1986). CDP IIb (195 kDa) is a homodimer, consisting of CalpM) a 95 kDa subunit, which appears to be the homolog of Cloning of a full-length cDNA sequence of Ha-CalpM from Drosophila CalpA or Dm-calpain (Beyette et al., 1993, 1997; Homarus americanus (Milne Edwards 1837) involved a three- Beyette and Mykles, 1997; Emori and Saigo, 1994; Jékely and step approach. First, a partial sequence within the protease Friedrich, 1999; Theopold et al., 1995). CDP IIb undergoes an domain of calpain was obtained with nested PCR. Second, autolytic cleavage at the N terminus, but it has little effect on inverse PCR (IPCR) was used to obtain the 5′ and 3′ ends of the Ca2+ sensitivity of the enzyme (Beyette et al., 1993; the sequence. Third, the full-length sequence was amplified by Beyette and Mykles, 1997). CDP IIa (125 kDa) is a homodimer PCR using primers derived from the IPCR sequence. In all of a 60 kDa subunit (Beyette et al., 1997). The subunit three steps, plasmid DNA (pcDNAII, Invitrogen) of a lobster compositions of CDP I (310 kDa) and CDP III (59 kDa) are not cDNA library made from deep abdominal muscle (Cotton and known, as neither has been purified to homogeneity. Mykles, 1993; Shean and Mykles, 1995) was used as template. Both Ca2+-dependent and ubiquitin/proteasome-dependent For the first step, the deduced amino acid sequences of proteolytic systems are stimulated during moult-induced claw calpain catalytic subunits from diverse species were aligned to muscle atrophy, when at least 40% of the muscle protein is identify conserved sequences using the Clustal W method from degraded (Beyette and Mykles, 1999; Mykles, 1998, 1999; the Lasergene biocomputing software (DNASTAR). Skinner, 1966). There is a preferential degradation of thin Degenerate primer sequences were selected from regions of myofilament proteins, resulting in a reduction in the high amino acid sequence identity. Two pairs of primers were thin:thick myofilament ratio and increased thick myofilament synthesized for nested PCR, covering a sequence segment of packing density (Ismail and Mykles, 1992; Mykles and 553 base pairs (bp) (equivalent to bp 423–975 in Drosophila Skinner, 1981, 1982). It is estimated that 11 thin myofilaments are degraded for every thick myofilament CalpA; accession number Z46891). The external primers were: ′ degraded (Mykles and Skinner, 1981). During postmoult, 1F (sense): 5 -C(T)TN GGN GAC(T) TGC(T) TGG C(T)TN ′ ′ protein is resynthesized as the muscle is restored to its C(T)T-3 and 1B (antisense): 5 -C(G)T(A) CAT CCA G(A)AA ′ ′ original mass and myofilament composition (Skinner, 1966). T(C)TC NCC G(A)TC-3 . The internal primers were: 1F ′ ′ Thus, there is significant remodeling of the contractile (sense): 5 -TAC(T) GCN GGN ATI TTC(T) CAC(T) TT-3 ′ ′ apparatus as fibers undergo atrophy and restoration during the and 1B (antisense): 5 -NCC CCA NGG G(A)TT NCT(G) ′ moulting cycle. Calpains probably play an important role, as NAT(A)(G) NC-3 . The primer locations are indicated in they degrade the Z-line and myofibrillar proteins in vitro and Fig. 1. in situ and have higher activities in atrophic muscle (Mattson The external primers were used for the first PCR reaction. and Mykles, 1993; Mykles, 1990; Mykles and Skinner, 1982, A Perkin-Elmer DNA thermal cycler model 9600 was used 1983). Moreover, the four calpain activities may have specific for all reactions. The composition of the reaction mixture µ –1 –1 functions, as each calpain has unique substrate specificities (25 l) was 2.5 mmol l MgCl2, 10 mmol l Tris-HCl, pH 9.0, –1 µ –1 when incubated with a mixture of myofibrillar proteins 50 mmol l KCl, 0.1% Triton X-100, 200 mol l each of µ –1 µ (Mattson and Mykles, 1993). For example, CDP IIb dNTPs, 1 mol l of each primer (1F and 1B), 0.75 g preferentially degrades thin myofilament proteins and thus plasmid, and 2.5 units of Taq polymerase (Promega). Initial may contribute to the loss of thin myofilaments in atrophic denaturation was at 94°C for 3 min, followed by 30 cycles of muscle (Mattson and Mykles, 1993). Proteasome level and denaturation at 94°C for 1 min, annealing at 54.8°C for 1 min, ubiquitin expression and conjugation are increased in and extension at 72°C for 1 min. The final extension was at atrophic muscle, although the role of this proteolytic pathway 72°C for 6 min. A 0.1 µl sample of the PCR reaction was used in claw muscle remains to be established (Koenders et al., as template for subsequent amplification with the internal 2002; Shean and Mykles, 1995). primers (1F′ and 1B′). The reaction conditions were the same Here we report the cloning of a crustacean calpain gene with as those for the first-round PCR. features that distinguish it from calpains isolated from other The Expand High Fidelity PCR System (Boehringer species. The cDNA encodes a non-EF-hand calpain with a Mannheim) was used for IPCR. Inverse primers were unique N-terminal sequence and an extended acidic loop in the synthesized based on the sequence of the nested PCR product: C2-like region of domain III. The transcript levels in various antisense, 5′-CAA GCC TTC CAT TGT ATG TG-3′ (bp tissues and in claw muscle during the moulting cycle were 600–619 in Fig. 1); sense, 5′-TCA TGC TTA CTC CAT CAC quantified by northern blot analysis, relative reverse CC-3′ (bp 909–928 in Fig. 1). The composition of the reaction transcription–polymerase chain reaction (RT-PCR) and real- mixture (50 µl) was 2 mmol l–1 Tris-HCl, pH 7.5, 10 mmol l–1 time PCR. The distribution of Ha-CalpM protein was KCl, 0.1 mmol l–1 dithiothreitol (DTT), 0.01 mmol l–1 EDTA, determined by western blotting and immunocytochemistry. 0.05% Tween-20, 200 µmol l–1 each of dNTPs, 300 nmol l–1 of Since this calpain was highly expressed in skeletal muscles, we each primer, 0.75 µg plasmid, and 2.6 units of Expand High have named the gene Ha-CalpM (Homarus americanus Fidelity PCR enzyme. Initial denaturation was at 94°C for muscle-specific calpain; GenBank accession no. AY124009). 2 min, followed by 10 cycles of denaturation at 94°C for 15 s, Muscle-specific calpain expression in Homarus americanus 563

aggagcaacaacaggATGTCGCTTAGTGAGGTGGTGGAGGATCAGAGTGTGTGGAAGCCAACGGAGGACCATGACAGTCACTGCCGCTGCTACAAACGGG 100 1 M S L S E V V E D Q S V W K P T E D H D S H C R C Y K R AGGGCCACGGCCACATGGTGGACGGCATAGAGTCCGTCTCCCTTCAGAAGTCTACCTACTACAGCCGCGTCAGTAATGATTACACCCAGAAGCGGATAGC 200 29 E G H G H M V D G I E S V S L Q K S T Y Y S R V S N D Y T Q K R I A CAAAGGTGGGTTGAAGATCCCCAAGAAGGGGTTCAGGACTCTGCGAGATGAGTGTCTTAAGAGCAGTAAGTTATACGAGGACCCTGAGTTCCCAGCCAAT 300 63 K G G L K I P K K G F R T L R D E C L K S S K L Y E D P E F P A N GACTACTCTATCAACTTCAATGGCGTCACCCGCAGAACCTATGTTTGGAAGAGACCTCACGAGATTACAAAGAATCCACGTTTCTTCATTGACGGTGCAA 400 96 D Y S I N F N G V T R R T Y V W K R P H E I T K N P R F F I D G A CACGGTTTGACATTCAACAGGGAGAACTTGGTGACTGTTGGCTCTTGGCTGCAGTATCTAATCTGACCTTGAATCCTCAAATGTTCCATGCGGTGGTACC 500 129 T R F D I Q Q G E L G D C W L L A A V S N L T L N P Q M F H A V V P AAGAGACCAAGGATTCATTGATCTTTATGCAGGCATCTTTCATTTCAAATTCTGGCAGTATGGGAAGTGGCAAGAGGTGGTGGTGGATGACAGGCTGCCC 600 163 R D Q G F I D L Y A G I F H F K F W Q Y G K W Q E V V V D D R L P ACATACAATGGAAGGCTTGTATTCATGCACTCAAAGACTGAAAATGAATTTTGGTGTGCTCTTTTTGAGAAGGCATATGCTAAGTTATATGGAGGGTATG 700 196 T Y N G R L V F M H S K T E N E F W C A L F E K A Y A K L Y G G Y AATCACTGCGTGGCGGCAACATTAATGAGAGTATGGTTGACCTGACAGGAGGTGTGGTGGAGATGATTGATCTTCGTAATCCACCACCCAAATTATTTTC 800 229 E S L R G G N I N E S M V D L T G G V V E M I D L R N P P P K L F S CACCTTGTGGAAGGCTTACCGTAGAGGTGCTCTCATAGGCACTGCTATAGAGCCCGACCAGTCAAACATACAGGCAGAGAGCATCCTCAGTAATGGCCTT 900 263 T L W K A Y R R G A L I G T A I E P D Q S N I Q A E S I L S N G L ATAATGCGTCATGCTTACTCCATCACCCGCGTTACTACAGTAGATATTAAGTCTGTTGTCCCCAGATTGCAGGGCAAGGCTCAACTAATCCGTCTGCATA 1000 296 I M R H A Y S I T R V T T V D I K S V V P R L Q G K A Q L I R L H ATCCTTGGGGCAATGAAGCTGAATGGAAAGGTTCGTGGAGTGACAAGTCTCCAGAGTGGAATTCTATCACTCCTGAGGAGAAGCAACGTCTTAAACTCAA 1100 329 N P W G N E A E W K G S W S D K S P E W N S I T P E E K Q R L K L N CTTTGAAGATGATGGGGAGTTTTGGATGAGTTTCCAGGACTTTGCATCAAATTTCACCACAGTTGAAATCTGTGATGTCACCCCAGAAGTGTTTGATCAT 1200 363 F E D D G E F W M S F Q D F A S N F T T V E I C D V T P E V F D H GATGATAGTGATGATGAAAATGGTAACACAAAGATGGAGGAGTCGGCACCAAAGAGGTGGCAGATGGTGATGTACGAAGGGGCCTGGGCTGCACACCATA 1300 396 D D S D D E N G N T K M E E S A P K R W Q M V M Y E G A W A A H H GTGCTGGTGGTTGCAGAAACTTTATTAACACATTTGCAAGCAACCCACAGTTTACAGTGCAGCTGGAGGATCCTGATGATGATGATGATGATGATGATGA 1400 429 S A G G C R N F I N T F A S N P Q F T V Q L E D P D D D D D D D D D TGATGACGATGATGATGATGATGACAGAGGTCAATGCACCATAGTTGTCTCTCTTATGCAGAAGAATGTCAGACAACTCAAACGCTATGGAGTTGACTAT 1500 463 D D D D D D D D R G Q C T I V V S L M Q K N V R Q L K R Y G V D Y GTCCCAATTGGCTTCACCATATATGCGTTGCCAGCAAACATGCAGCCTGGACAAAAGCTGGACACAGAGTTCTTCAAGTACAATCCTAGCTTGGCAAAAG 1600 496 V P I G F T I Y A L P A N M Q P G Q K L D T E F F K Y N P S L A K TACCATTTTTCCTCAATACTCGTGAATTAACTACTAGATTCCGTTTCCCTCCTGGTCTTTATGTCATCATCCCATCCACTTTTGAACCTGAAATGACTG 1700 529 V P F F L N T R E L T T R F R F P P G L Y V I I P S T F E P E M T GAGAGTTTCTCATAAGAATCTTTACTGAGGCTAAGAGAAGGTAAattgtaattatttttaagtgacatattaagtctcaggatttagcaacaaacgcatg 1800 562 G E F L I R I F T E A K R R STOP acaaaatactactgtacactatcaaatagatttgtgttttatagaattttatcttaattatttactgtacaagtaaaagcaaatgagacagtgaaaatat 1900

ttgtataccggtacctttaaagtgaataacttttattcaagaagacagtaattagaatgctaatgatgggctgtgcaa 1977

Fig. 1. The complete sequence of Ha-CalpM cDNA cloned from a lobster fast muscle library. The sequence (1977 bp) contains an open reading frame (bp 16–1743) encoding a protein of 575 amino acids (aa) with a predicted mass of 66.3 kDa. The three conserved aa (Cys 141, His 299, Asn 329) essential for hydrolase activity are indicated in bold. A stretch of 17 aspartates (aa 454–470) is underlined. The numbers on the left indicate aa positions; those on the right indicate nucleotides. The highly conserved aa sequences used for initial cloning with nested PCR are underlined and italicized. GenBank accession no. AY124009. annealing at 59°C for 30 s, and extension at 68°C for 4 min. Positive colonies were selected and grown overnight; plasmid The reaction then underwent 20 cycles of denaturation at 94°C DNA was isolated using a Qiagen Plasmid Mini-Prep kit. for 15 s, annealing at 55°C for 30 s and extension at 68°C for Plasmid DNA containing inserts were sequenced in both 4 min. The final extension was at 72°C for 6 min. directions using the promoter primers Sp6 and T7 and gene- Based on the sequence from IPCR, specific primers to the specific primers (Davis Sequencing, Davis, CA, USA). 5′ and 3′ ends of Ha-CalpM were synthesized: sense, 5′-AGG For sequence analysis, calpain sequences were retrieved AGC AAC AAC AGG ATG-3′ (bp 1–18 in Fig. 1); antisense, from GenBank at the National Center for Biotechnology 5′-TGC ACA GCC CAT CAT TAG –3′ (bp 1959–1976 in Fig. Information (NCBI) (http://www.ncbi.nlm.nih.gov). Sequence 1). The PCR conditions were the same as those used for IPCR. homologies were determined using the NCBI BLAST For cloning PCR products, reactions were separated program. Multiple alignment and phylogenetic analyses were electrophoretically on 1% agarose gels (70 V for 1.5 h); done using DNASTAR sequence analysis software. products were excized and purified using the Qiagen Gel Purification kit. The DNA was concentrated in a Savant Analysis of Ha-CalpM mRNA by northern blot and RT-PCR Speedvac and ligated into the pGEM-T Easy plasmid vector Total RNA from lobster tissues was extracted using Trizol (Promega). Ligation was achieved by incubating 250 ng DNA, reagent (Gibco BRL). The procedure was essentially the same 50 ng vector and 3 Weiss units T4 DNA ligase in 30 mmol l–1 as that recommended by the manufacturer. Briefly, tissues were –1 –1 Tris-HCl, pH 7.8, 10 mmol l MgCl2, 10 mmol l DTT, homogenized in 10 vol. Trizol (500 mg tissue in 5 ml) using a 0.5 mmol l–1 ATP and 5% polyethylene glycol, at room Polytron homogenizer (Brinkmann Instruments, Westbury, temperature for 1 h or overnight at 4°C (10 µl final volume). NY, USA) for about 1 min or until the tissues were completely DH5α electro-competent E. coli cells were transformed with homogenized. Insoluble material was removed by the ligation reaction by electro-transformation and plated on centrifugation at 12 000 g for 10 min at 4°C. 1 ml chloroform LB agar plates containing 100 µgml–1 ampicillin (Sigma). was added to each supernatant fraction after incubation at room 564 X. Yu and D. L. Mykles temperature for 5 min. Samples were vortexed and centrifuged Fig. 1). The reaction mixture contained 1× ExTaq buffer at 12 000 g for 15 min at 4°C. RNA in the aqueous phase was (2.5 mmol l–1 Mg2+, 200 µmol l–1 dNTP, 0.5 µmol l–1 of each precipitated with 2.5 ml isopropyl alcohol and pelleted by primer, 1.5 units of Hot Start ExTaq polymerase (Takara). The centrifugation at 12 000 g for 10 min at 4°C. The precipitated final volume was 25 µl. After synthesis of first-strand cDNA RNA was washed with 75% ethanol, dissolved in 50 µl (see above), PCR amplification followed at 92°C for 2 min, and deionized formamide, and stored at –80°C. 30 cycles of 94°C for 30 s, 60°C for 30 s and 68°C for 45 s. Total RNA (20 µg) was separated electrophoretically on 1% The final extension time was 6 min at 72°C. The conditions for agarose gels containing 0.5 mol l–1 formaldehyde in 1× Mops amplifying lobster α-actin (Ha-actin1; accession no. buffer (40 mmol l–1 Mops, pH 7.0, 10 mmol l–1 sodium acetate AF399872) were the same as that for Ha-CalpM and used the and 1 mmol l–1 EDTA) at 70 V for 3 h. RNA was transferred same primers described previously (Koenders et al., 2002). onto Hybond-N nylon membrane (Amersham) overnight in Real-time PCR quantification of Ha-CalpM transcript in 20× SSC (3 mol l–1 NaCl and 0.3 mol l–1 sodium citrate, lobster tissues used a SmartCycler instrument (Cepheid). A pH 7.0). The blot was UV cross-linked at 120 J for 30 s PCR master mix was assembled on ice containing 1× and hybridized in 20 ml hybridization buffer (Boehringer LightCycler FastStart Reaction Mix (SYBR Green I, dNTP Mannheim) containing 50% deionized formamide, 5× SSC, mix with dUTP instead of dTTP, and FastStart Taq DNA –1 –1 0.1% N-lauroylsarcosine, 0.02% SDS, 1% Blocking Reagent, polymerase), 3 mmol l MgCl2 and 0.5 µmol l of each primer and 1 µg digoxigenin (DIG)-labeled RNA probe overnight at (20 µl total volume). The sense primer (RTCalp-F1) was 5′- 68°C. A DIG-RNA Labeling kit (Boehringer Mannheim) was CAA CAA CAG GAT GTC GCT TAG TGA G-3′ (bp 6–30 used to synthesize the calpain probe by in vitro transcription in Fig. 1) and the antisense primer (RT-Calp-B1) was 5′-TTC from linearized plasmid containing a partial Ha-CalpM insert TGA AGG GAG ACG GAC TCT ATG-3′ (bp 126–149 in Fig. (bp 646–1129 in Fig. 1). Plasmid (1.8 µg) was linearized 1). The mixtures were placed in reaction tubes, followed by the by digestion with NcoI (37°C for 1 h), separated addition of 1 µl (100 ng) of first-strand cDNA. Plasmid electrophoretically on a 1% agarose gel, and purified with a containing Ha-CalpM cDNA insert was serially diluted (every Qiagen gel purification kit. The labeling reaction mixture tenfold) at a range of 0.28 fg to 28 pg to generate a standard (20 µl) contained 0.78 µg linearized plasmid, 1× DIG-RNA curve. Both the Ha-CalpM plasmid and first-strand cDNA labeling mix (1 mmol l–1 ATP, 1 mmol l–1 CTP, 1 mmol l–1 reactions were run simultaneously in a SmartCycler instrument GTP, 0.65 mmol l–1 UTP and 0.35 mmol l–1 DIG-11-UTP, using the same protocol. The protocol consisted of three stages: pH 7.5), 1× transcription buffer, pH 7.5, and 30 units Sp6 RNA stage 1, holding at 95°C for 10 min; stage 2, 40 cycles at 95°C polymerase; the mixture was incubated at 37°C for 2 h. The for 10 s, 57°C for 15 s with Optics on, and 72°C for 10 s; and membrane was washed twice (1 h each) with 0.5× SSC stage 3, melting curve (60°C to 95°C at 0.2°C s–1). Cycle and 0.1% SDS at 68°C to remove unhybridized probe. threshold analysis was used to quantify the absolute copy Chemiluminescent detection of probe used anti-DIG antibody number of the Ha-CalpM transcript, as the cycle at which a conjugated to alkaline phosphatase (Boehringer Mannheim) specific PCR product begins to accumulate is directly and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′- proportional to the concentration of cDNA template in the (5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate reaction. A standard curve plotting the cycle threshold versus (CSPD; Roche Molecular Biochemicals) according to Ha-CalpM cDNA concentration was used to calculate the Boehringer Mannheim’s protocol. concentration of Ha-CalpM transcript in first-strand cDNA For first-strand cDNA synthesis, total RNA in formamide synthesized from tissue RNA. was precipitated with 0.1 vol. 4 mol l–1 LiCl and 3 volumes of 100% ethanol. The RNA was washed in 75% ethanol and Purification and N-terminal sequencing of Ha-CalpM dissolved in diethyl pyrocarbonate (DEPC)-treated H2O. RNA isoforms was first treated with DNase (Gibco BRL) to remove Chromatographic isolation of Ha-CalpM followed the contaminating DNA, then 2 µg of RNA was reverse- protocol described previously (Mykles, 2000). Briefly, claw transcribed using SuperScriptII RNase H– reverse and deep abdominal muscles were homogenized in Buffer A transcriptase (Gibco BRL). The reaction consisted of 2.5 µg of (20 mmol l–1 Tris-acetate, pH 7.5, 20 mmol l–1 KCl, 1 mmol l–1 Oligo (dT)12–18 (or 0.5 µg of random primers), 2.5 mmol l–1 EDTA and 1 mmol l–1 DTT) and centrifuged at 12 000 g for dNTP, 1× First-Strand Buffer, 5 mmol l–1 DTT, 2.5 units 15 min at 4°C. The supernatant was precipitated with 65% RNase inhibitor and 200 units Superscript II RNase H– saturated ammonium sulfate, dialyzed overnight against 2 l Reverse Transcriptase. The reaction mixture was incubated at Buffer A without DTT and KCl at 4°C, and chromatographed 42°C for 50 min and stored at –20°C. on an organomercurial-agarose (OMA) column (5 cm × 5 cm). For the relative RT-PCR, a pair of 18S rRNA Primers and Active fractions from the OMA column were concentrated by Competimers of ratio 3:7 (Ambion) were included in the PCR a stirred-cell ultrafiltration device (Model 8200, Amicon) and reaction mixture together with the calpain primer pairs directed dialyzed against two 2 l changes of Buffer A. Protein was to the 5′ end of the Ha-CalpM cDNA: sense, 5′-AGG AGC loaded onto a Q Sepharose column (2.5 × 18 cm) and eluted AAC AAC AGG ATG-3′ (bp 1–18 in Fig. 1); antisense, 5′- with a linear NaCl gradient from 0 to 1 mol l–1 in Buffer A CAA GCC TTC CAT TGT ATG TG-3′ (bp 600–619 in (300 ml total volume). Western blotting (see below) was used Muscle-specific calpain expression in Homarus americanus 565 to identify Ha-CalpM-containing fractions, which were pooled, Immobilon-AV membrane was incubated with 1 mg peptide concentrated to about 5 ml with Filtron Macrosep 50K devices, in 2 ml 0.5 mol l–1 potassium phosphate, pH 7.4, at room and dialyzed against 2 l Buffer B (20 mmol l–1 Pipes-NaOH, temperature for 2 h. The membrane was washed with 1 mol l–1 –1 –1 pH 6.8, 0.1 mol l NaCl, 1 mmol l EDTA, 20% glycerol and NaHCO3 for 2 h, followed by two 30 min washes in PBS 1 mmol l–1 DTT). The dialyzed sample was chromatographed (phosphate-buffered saline; 80 mmol l–1 disodium hydrogen on a Superdex 200 (Pharmacia) gel filtration column (2.6 cm × orthophosphate, 20 mmol l–1 sodium dihydrogen 86 cm) equilibrated with Buffer B at 3 ml min–1. Fractions orthophosphate and 100 mmol l–1 NaCl, pH 7.5) containing (1.5 ml) containing Ha-CalpM (determined by western 0.1% Tween-20. The membrane was incubated with 2 ml of the blotting) were pooled and loaded onto a Mono Q ion exchange Ha-CalpM antiserum for 3.5 h, rinsed three times with PBS, column (Pharmacia HR 5/5) equilibrated with Buffer B. and bound antibody was eluted in 0.9 ml 100 mmol l–1 glycine, Protein was eluted from the column with a linear NaCl gradient pH 2.5, for 10 min. The solution was neutralized with 0.1 ml from 0.1 to 0.5 mol l–1 (20 ml total volume) at 1 ml min–1. 1 mol l–1 Tris-HCl, pH 8.0, and stored at 0°C with 0.02% Fractions (0.6 ml) were analyzed by western blotting using Ha- sodium azide. The concentration of immunoglobulin G (IgG) CalpM antiserum. was determined by dot-blot analysis comparing serial dilutions For N-terminal sequencing, the Ha-CalpM proteins of affinity-purified IgG with serial dilutions of known (Mono Q fractions 23–25) were separated on a 6% SDS- concentrations of rabbit IgG; detection used the Biotin/Avidin polyacrylamide gel and transferred to PVDF (polyvinylidene System (see below). difluoride) membrane using 1× CAPS, pH 11/10% methanol The detection of Ha-CalpM used the Biotin/Avidin System transfer buffer. The protein on the blot was stained with India (Vector Laboratories). The membrane was blocked with 5% ink and the 62 kDa and 68 kDa proteins were excized and non-fat milk in TBS (Tris-buffered saline; 20 mmol l–1 Tris- submitted to the Colorado State University Macromolecular HCl, pH 7.5, and 137 mmol l–1 NaCl) at room temperature for Resources Facility for sequencing by Edman degradation. 1 h. The primary antibody incubation was 1 h with Ha-CalpM antiserum or affinity-purified IgG (1:10,000 dilution) in 3% SDS-polyacrylamide gel electrophoresis and western blot non-fat milk in TBS-T (0.01% Tween-20 in TBS). The analysis secondary antibody incubation was 1 h with biotinylated goat For analysis of Ha-CalpM in lobster tissues, tissues were anti-rabbit IgG (1:10,000 dilution in TBS-T). The membrane homogenized in 6 vol. ice-cold Buffer A containing 1× was incubated with ABC solution (10 µl avidin and 10 µl protease inhibitor cocktail {Sigma; 2 mmol l–1 AEBSF [4- biotinylated horseradish peroxidase in 10 ml TBS-T) for (2-aminoethyl)benzene sulfonyl fluoride], 1 mmol l–1 EDTA, 30 min. ECL Plus (Amersham Pharmacia) was used for 130 µmol l–1 bestatin, 1.4 µmol l–1 E-64, 1 µmol l–1 leupeptin chemiluminescent detection of peroxidase activity. and 0.3 µmol l–1 aprotinin} and centrifuged at 12,000 g for 15 min at 4°C. The supernatant fractions were removed and Immunocytochemistry stored on ice. Protein concentration was determined by Tissues were fixed in 4% paraformaldehyde in lobster fluorescence emission with a Perkin-Elmer LS-2 Filter saline (0.5 mol l–1 NaCl, 15 mmol l–1 KCl, 10 mmol l–1 Fluorimeter using bovine serum albumin as standard (Avruch EDTA, 25 mmol l–1 Hepes-NaOH, pH 7.5), dehydrated and Wallach, 1971). through an ethanol series, and embedded in paraffin. Protein samples (32 µg) were mixed with equal volumes of Immunodetection used the Vectastain ABC Elite kit (Vector sample buffer (150 mmol l–1 Tris-HCl, pH 6.8, 2% SDS, 10% laboratories) with minor modifications (Mykles et al., 2000). glycerol, 20 mmol l–1 DTT and 0.01% Bromophenol Blue) and After hydration, sections (10 µm) were washed twice in PBS incubated for 5 min at 95°C. Proteins were separated buffer containing 15 mmol l–1 glycine (PBS/Gly) for 5 min electrophoretically by discontinuous SDS-polyacrylamide gel and blocked with 1.5% normal goat serum in PBS/Gly for 1 h. electrophoresis (SDS-PAGE; Laemmli, 1970). For the column Sections were incubated with Ha-CalpM antiserum or pre- chromatography samples, 10 µl of each fraction was analyzed. immune serum (1:5000 dilutions in 1.5% normal goal serum Proteins in gels were either stained with ammoniacal silver in PBS/Gly) overnight. After washing with PBS/Gly, sections (Mykles, 1988) or electrophoretically transferred to PVDF were incubated with biotinylated goat anti-rabbit IgG membrane (Hybond-P, Amersham) (Towbin et al., 1979). (1:200 dilution in 1.5% normal goat serum in PBS/Gly) for For immunochemical studies, a polyclonal antibody was 2 h, followed by incubation with ABC reagent (avidin/ raised against a 28-amino acid (aa) sequence (53–80) in the N- biotinylated horseradish peroxidase complex) for 30 min. terminal region of the Ha-CalpM deduced sequence. Peptide Antibody was detected colormetrically by incubating –1 (NH2-SNDYTQKRIAKGGLKIPKKGFRTLRDEC-COOH) the sections in a solution containing 0.8 mg ml conjugated to keyhole limpet hemocyanin was used to raise diaminobenzidine tetrahydrochloride, 0.4 mg ml–1 NiCl, –1 antibody in rabbits by the Macromolecular Resource Facility 0.01% H2O2, and 0.1 mol l Tris-HCl, pH 7.4. The sections at Colorado State University. were rinsed with deionized H2O, dehydrated through an In some cases anti-Ha-CalpM IgG was affinity-purified ethanol series, cleared in xylene, and mounted in Permount using the Ha-CalpM peptide covalently-bonded to Immobilon- (Fisher Scientific). Sections were photographed under bright- AV membrane (Millipore). Briefly, a 2 cm × 2 cm square of field illumination. 566 X. Yu and D. L. Mykles

Fig. 2. Comparison of Ha-CalpM amino acid (aa) sequence with calpains from Drosophila and human. The deduced aa sequence of Ha-CalpM was aligned with Drosophila (CalpA and CalpB) and human (m-Calp) calpain sequences (see Materials and methods). Gaps (broken lines) were introduced to optimize the alignment. Aa residues that are identical to the Ha-CalpM sequence are highlighted. Ha-CalpM is highly homologous to Drosophila calpains (50% identity, 67% similarity). Ha-CalpM has three domains: domain I (aa 1–110) has a unique N-terminal sequence comprising the ﬁrst 75 aa residues, domain II (aa 111–386) consists of a highly conserved cysteine proteinase sequence and domain III (aa 387–575) has a C2-like region that includes an acidic loop containing a stretch of 17 aspartate residues (aa 454–470). Ha-CalpM lacks a calmodulin-like domain (Domain IV) found in the human m-calpain and Drosophila sequences. Ha-CalpM has a short insertion sequence (aa 398–407) in domain III near the boundary with domain II that is not found in the other sequences. The boundaries between domains I and II, II and III, and III and IV are indicated by I/II, II/III and II/IV, respectively. The open triangles indicate the three amino acid residues of the catalytic triad (Cys141, His299, Asn329) in domain II. The asterisks indicate two conserved aspartate residues (Asp132 and Asp366) in domain II that bind Ca2+. Domain IV in the Drosophila and human sequences contains ﬁve EF-hand motifs (EF-1, EF-2, etc.). GenBank accession numbers are Z46891 for Drosophila calpain A (CalpA), AF062404 for Drosophila calpain B (CalpB), and M23254 for human m-calpain (m- Calp).

Modeling of lobster calpain conserved amino acid sequences in the calpain proteinase The Ha-CalpM sequence (aa residues 61–575) was modeled domain yielded five partial cDNAs comprising three distinct using the crystal structure of mammalian m-calpain as template sequences, all of which revealed high identities with members (Hosfield et al., 1999; Strobl et al., 2000). Swiss-Model and of the calpain gene family (data not shown). One of the Sybyl programs were used for initial tracing of the backbone. sequences belonged to Ha-CalpM; the sequences of the other Further refinement of the putative secondary structure used the clones will be submitted once full-length sequences are algorithm from Kabsch and Sander (1983) using UCSF obtained (X. Yu, H. W. Kim and D. L. Mykles, unpublished MidasPlus software with these parameters: energy cutoff for data). In order to obtain a full-length sequence, inverse PCR hydrogen bonding, –0.5 kcal mol–1; minimum helix length, 3; (IPCR) was used to amplify the sequences flanking the initial minimum sheet length, 3. PCR products. Since the templates were plasmid DNA from a lobster deep abdominal muscle cDNA library, the expected size of the IPCR product would be the length of the calpain Results gene plus the vector (3 kb). A 5 kb product was amplified, Characterization of Ha-CalpM cDNA cloned, and partially sequenced (data not shown). The end Nested PCR using degenerate primers directed to four highly sequences of Ha-CalpM were located and primers synthesized. Muscle-specific calpain expression in Homarus americanus 567

Fig. 3. Phylogenetic relationship of Ha- Dm-CalpA-II CalpM to other calpains. Amino acid Dm-CalpB-II Ha-CalpM-II sequences of the proteinase domain human p94-II (domain II) of calpains from selected nCL-4-II species were compared using the Clustal human m-II Capn5 (mouse)-II V method (see Materials and methods). tra-3-II Branch lengths are proportional to the Shistosoma-II 26.7 inferred phylogenetic distances, with the Shistosoma mansoni domain II sequence 25 20 15 10 5 0 serving as the outgroup. Ha-CalpM was grouped with Drosophila calpains (Dm-CalpA and Dm-CalpB), reﬂecting the high sequence identity among the three sequences. GenBank accession numbers for Drosophila calpains and human m-calpain are given in the legend of Fig. 2; the accession numbers for the other calpains are X92523 for human p94 (CAPN3), AF022799 for mouse nCL-4 (CAPN9), M74233 for S. mansoni, Y10656 for mouse calpain 5 (CAPN5), and U14480 for C. elegans tra-3.

The final full-length cDNA was PCR-amplified with the 3′ and 5′ end primers and sequenced. The full-length Ha-CalpM cDNA consists of 1977 nucleotides (Fig. 1). The position of the initiation codon is similar to that of Drosophila calpains (Jékely and Friedrich, 1999; Theopold et al., 1995). The Ha-CalpM cDNA has a 5′ UTR of 15 bp and a 3′ UTR of 234 bp. The open reading frame (ORF; bp 16–1743) encodes a protein of 575 amino acids with a predicted molecular mass of 66.3 kDa. Alignment of the deduced aa sequence of Ha-CalpM with Drosophila and human calpains is shown in Fig. 2. Ha-CalpM has the highest sequence identity with Drosophila calpains A and B (CalpA and CalpB; 50% identity, 67% similarity). In addition to Drosophila calpains and human m-calpain, Ha- CalpM also has significant sequence similarities to other mammalian calpains, such as digestive tract-specific calpain (CAPN9 or nCL-4; AF022799; 64% similarity) and skeletal Fig. 4. Northern blot analysis of Ha-CalpM expression in lobster muscle-specific calpain (CAPN3 or p94; AF127765; 62% tissues. Total RNA (20 µg) was separated electrophoretically on a similarity). The similarity to human m-calpain is 60%. The 1% agarose gel and blotted overnight. The membrane (A) was domain organization of Ha-CalpM was assigned according to hybridized under high stringency with a DIG-labeled RNA probe that of the Drosophila calpain sequences (Sorimachi and synthesized from a plasmid with an insert encoding the region Suzuki, 2001). Domain I (aa 1–110) has a unique N-terminal encompassing amino acid residues 171–332 in domain II of Ha- sequence (aa 1–75) that has no sequence identity with any other CalpM (Fig. 1; see Materials and methods). Three Ha-CalpM sequence in the NCBI database. Domain II (aa 111–396) transcripts were detected (3.3, 4.0 and 6.1 kb) in cutter (Cu) and consists of a highly-conserved cysteine proteinase sequence crusher (Cr) claw muscles and deep abdominal (DA) muscle containing the catalytic triad (Cys141, His299 and Asn329) and (A, lanes a–c). The fast muscles expressed all three mRNAs (lanes a and c), while the slow muscle (lane b) expressed primarily the 3.3 kb two aspartates (Asp132 and Asp366) involved in Ca2+ binding. mRNA. Intestine (In; lane f) showed a strong hybridization Domain III (aa 397–575) contains a short insertion sequence to mRNA in the 3.3–6.1 kb range, as well as to mRNAs greater than (aa 408–417) and an acidic loop with an expanded stretch of an 6.1 kb. Little or no Ha-CalpM mRNA was detected in digestive gland 2+ additional 11 aspartates (aa 454–470) that may bind Ca and (DG; lane d), gill (Gi; lane e), nerve cord (NC; lane g), integument help activate the proteinase. Ha-CalpM lacks domain IV (a (Ig; lane h), heart (Ht; lane i) or antennal gland (AG; lane j). RNA calmodulin-like domain) found in EF-hand calpains. loading was determined by staining the gel with Ethidium Bromide A phylogenetic tree was generated comparing the domain II (B). Lane M, molecular mass markers (kb) are indicated. amino acid sequence of Ha-CalpM (Ha-CalpM-II) with the domain II sequences of selected calpains representing diverse phyla (Fig. 3). The Shistosoma calpain domain II sequence Expression of Ha-CalpM served as the outgroup in the analysis. As expected, Ha-CalpM Northern blotting was used initially to determine the tissue clustered with Drosophila CalpA and CalpB. The human p94 distribution and sizes of Ha-CalpM mRNAs. The membrane (Capn3), human m-calpain (Capn2), and mouse nCL-4 was hybridized with a DIG-labeled RNA probe synthesized (Capn9) formed a distinct, but related group; mouse Capn5 and from the domain II region (equivalent to aa 171–332) of Ha- C. elegans tra-3 calpains were more divergent. CalpM under high stringency. The results show that Ha-CalpM 568 X. Yu and D. L. Mykles

Fig. 5. RT-PCR analysis of Ha-CalpM and α-actin expression in lobster tissues. Total RNA from various tissues were DNase-treated, reverse-transcribed, and PCR-amplified using primers directed to the 5′ ends of Ha-CalpM (bp 1–18 and 600–619 in Fig. 1) and Ha- Actin1 (bp 1–23 and 379–400; see Koenders et al., 2002). PCR amplification of 18S rRNA served as an internal standard. (A) The PCR products synthesized from Ha-CalpM (619 bp) and 18S rRNA (324 bp). (B) The PCR product (400 bp) synthesized from α-actin. Shown are negative images of ethidium bromide-stained agarose gels. There were high amounts of Ha-CalpM mRNA in cutter claw, crusher claw and deep abdominal muscles (lanes a–c). Little or no Ha-CalpM product was amplified from other tissues (lane e–i). α- Actin was expressed in all tissues except antennal gland (lane i). Abbreviations as in Fig. 4.

Table 1. Quantification of Ha-CalpM transcript in lobster tissues Fig. 6. SDS-PAGE and western blot analysis of Ha-CalpM in lobster Number of transcripts ng–1 tissues. Supernatant fractions (32 µg protein) from various tissues Tissue total RNA% were electrophoresed on 6% SDS-polyacrylamide gels and either Deep abdominal muscle 6753 100 stained with Coomassie Blue (C) or transferred onto PVDF Cutter claw muscle 6570 97 membrane and probed with affinity-purified anti-Ha-CalpM IgG (A) Crusher claw muscle 1612 24 or pre-immune IgG (B). The Ha-CalpM antibody detected a 62 kDa Integument 183 3 isoform in claw muscles (lanes a and b) and a 68 kDa isoform in Gill 154 2 deep abdominal muscle (lane c). Non-specific binding of the Digestive gland 127 2 antibody (B, lanes e–g) is indicated by asterisks in A. Hemocyanin, Nerve cord/thoracic ganglion 88 1 an extracellular 78 kDa oxygen-binding protein in the hemolymph, is Intestine 85 1 a major contaminant in all tissues. Positions of molecular mass Antennal gland 29 0.4 standards (kDa) are indicated. Te, testis; all other abbreviations as in Fig. 4. mRNA was reverse-transcribed and amplified by real-time polymerase chain reaction. Reactions containing known RT-PCR was used to detect low levels of expression and to concentrations of lobster CalpM cDNA were used to generate a determine whether the hybridization of the probe to intestine standard curve to quantify CalpM mRNA (see Materials and methods). RNA (Fig. 4) was specific to Ha-CalpM. Primers were Ha-CalpM mRNA levels were approximately fourfold greater in synthesized to amplify the sequence encoding the unique N- fast skeletal muscle (deep abdominal and cutter claw muscles) than terminal sequence of Ha-CalpM. As expected, a large amount in slow skeletal muscle (crusher claw muscle). Other tissues of PCR product (619 bp) was synthesized from muscle RNAs expressed Ha-CalpM at much lower levels. (Fig. 5, lanes a–c). Little or no Ha-CalpM PCR product was obtained from digestive gland, gill, intestine, nerve cord/ thoracic ganglion, integument and antennal gland (Fig. 5, was highly expressed in skeletal muscle (Fig. 4, lanes a–c). lanes d–i), indicating that the hybridization in the northern blot Two major transcripts (6.1 kb and 3.3 kb) were detected. A to intestine RNA was not due to high levels of Ha-CalpM minor 4 kb mRNA was also present in fast muscles (cutter claw mRNA. PCR amplification of 18S rRNA (324 bp) and α-actin and deep abdominal). The probe hybridized to mRNAs of (Ha-Actin1; 400 bp) products indicated that each lane varying sizes from intestine (Fig. 4, lane f). contained the same amount of total RNA and that the RNA Muscle-specific calpain expression in Homarus americanus 569

Fig. 7. Immunocytochemical localization of Ha-CalpM in claw muscles from adult lobster. Transverse sections were stained with Hematoxylin/Eosin (B) or incubated with pre-immune serum (A) or Ha-CalpM antiserum (C,D). Nuclei (arrowheads) are associated with the sub-sarcolemmal cytoplasm of sarcolemal clefts (arrows), which are large infoldings of the cell membrane. The Ha-CalpM antiserum stained the cytoplasm uniformly and some nuclei. Each field of view contains portions of two muscle fibers. ES, extracellular space. Scale bar, 100 µm. was not degraded (Fig. 5). A negative PCR control using total crusher claw muscles showed that Ha-CalpM was distributed RNA as template produced no products, indicating no DNA throughout the cytoplasm (Fig. 7). Ha-CalpM was also contamination (data not shown). The α-actin was expressed at localized in some, but not all, nuclei (Fig. 7, compare the a higher level in the crusher claw muscle than in the cutter Hematoxylin staining of nuclei in B with the staining of fewer claw and deep abdominal muscles, which corresponds to the nuclei in C and D). higher ratio of actin to myosin in slow fibers than in fast fibers As calpains are involved in moult-induced claw muscle (Mykles, 1985, 1988). The same RNA was used to quantify atrophy, the expression of Ha-CalpM was determined in claw the copy number of Ha-CalpM mRNA in the tissues by real- muscle from lobsters at different moult stages. For real-time time PCR (Table 1). The highest copy numbers were in PCR, total RNA was isolated from crusher claw muscle at skeletal muscles, although the amount in slow muscle (crusher intermoult, early premoult (D0), and postmoult (2–4 days post- claw) was about 25% that in fast muscle (cutter claw and deep ecdysis) stages, reverse-transcribed and PCR-amplified in the abdominal). Other tissues expressed Ha-CalpM at much lower presence of SYBR Green I fluorescent dye (Fig. 8). There was copy numbers (0.4% to 3% that of deep abdominal muscle; no significant effect of moult stage on Ha-CalpM mRNA levels Table 1). (P<0.28; ANOVA single factor analysis). Parallel PCR An antibody raised against a 28-aa sequence in the N- reactions using Ha-actin1 primers showed no changes in Ha- terminal domain of Ha-CalpM (aa 53–80) was used to actin1 mRNA (data not shown), which was consistent with the determine Ha-CalpM protein levels and distribution in lobster constitutive expression of actin during the moutling cycle tissues. In western blots, the antibody detected two proteins reported recently (Koenders et al., 2002). The 62 kDa Ha- in skeletal muscles: a 62 kDa isoform in cutter and crusher CalpM isoform was expressed in claw muscles at all stages of claw muscles and a 68 kDa isoform in deep abdominal the moulting cycle (Fig. 9). As levels were quite variable in muscle (Fig. 6, lanes a–c). The isoforms were not detected intermoult muscle (Fig. 9, lanes a,b), the higher levels of Ha- in gill, intestine, heart or testis (Fig. 6, lanes d–g). CalpM in postmoult claw muscles (lanes e,f) were not Immunocytochemistry of transverse sections of the cutter and significantly different. 570 X. Yu and D. L. Mykles

10000 RNA

–1 1000

100

Number of copies ng 1 Intermoult Premoult Postmoult Fig. 8. Real-time PCR analysis of Ha-CalpM expression in lobster crusher claw muscles during the moulting cycle. Total RNA from intermoult, early premoult (stage D0), and early postmoult (2–4 days post-ecdysis) lobsters was DNase-treated, reverse-transcribed, and PCR-amplified in reactions containing SYBR Green I fluorescent dye (see Materials and methods). Values are means ±1 S.D.(N=4 for each moult stage). Ha-CalpM transcript copy number did not change significantly (P<0.28; ANOVA single factor analysis). Fig. 9. Western blot analysis of Ha-CalpM in claw muscles during Purification of Ha-CalpM the moulting cycle. Supernatant fractions (32 µg protein) of claw muscles at different moulting stages were electrophoresed on 6% Four calpain activities (CDPs I, IIa, IIb and III) have been SDS-polyacrylamide gels and either stained with Coomassie Blue characterized biochemically in lobster skeletal muscles (B) or transferred onto PVDF membrane and probed with Ha-CalpM (Mykles and Skinner, 1986). The mass of the deduced Ha- antiserum (A) or pre-immune serum (data not shown). The 62 kDa CalpM sequence (66.3 kDa) and masses of the Ha-CalpM isoform (arrow) was present at all moulting stages: intermoult (lanes isoforms in western blots (62 kDa and 68 kDa) suggested that a,b), premoult (lanes c,d), and postmoult (lanes e,f). Proteins binding Ha-CalpM cDNA encoded either CDP IIa (60 kDa subunit non-specifically to the antibody are indicated by asterisks in A. mass; 125 kDa native mass) or CDP III (59 kDa native mass). Positions of molecular mass standards (kDa) are indicated. I-1 and I- A soluble extract of intermoult lobster muscles (claws and 2, intermoult stage at 20% and 30% of the intermoult interval, deep abdominal) containing all four calpain activities was respectively; D1, premoult stage D1; D3, premoult stage D3; P-1 and subjected to a series of chromatographic separations on P-2, postmoult stage at 7% and 13% of the intermoult interval, organomercurial agarose, Q Sepharose, Superdex-200 gel respectively. filtration and Mono Q columns (Mykles, 2000). The 62 kDa and 68 kDa Ha-CalpM isoforms co-eluted from each column (Herasse et al., 1999; Theopold et al., 1995) or domain II and the results are shown for the final two separations (Figs 10, (Sorimachi et al., 1993). It is unlikely that Ha-CalpM is a 11). The Ha-CalpM isoforms eluted from the Superdex-200 gel truncated form of a longer calpain sequence homologous to filtration column at a position corresponding to the native mass Dm-CalpA. The antibody to the N-terminal domain reacts only of CDP III (Fig. 10, lanes h–n). The gel filtration fractions with the 62 kDa and 68 kDa isoforms; there is no other protein containing Ha-CalpM were concentrated and chromatographed detected in soluble extracts of skeletal muscle or any other in a Mono Q anion exchange column; the Ha-CalpM proteins tissue (Fig. 6). eluted as a single peak (fractions 23–25; Fig. 11, lanes i,j). It is likely that Ha-CalpM has Ca2+-dependent proteinase Edman degradation of the purified 62 kDa and 68 kDa isoforms activity, even though it lacks domain IV. Ca2+-binding regions did not yield N-terminal sequences for either polypeptide. in domains II and III in other calpains (Andresen et al., 1991; Hosfield et al., 2001; Moldoveanu et al., 2002) are highly conserved in Ha-CalpM (Fig. 2). Expressed recombinant Discussion mammalian calpains lacking domain III or IV or only Ha-CalpM is the first crustacean calpain gene that has been containing domain II retain Ca2+ dependency (Hata et al., cloned and sequenced. It is classified as a non-EF-hand calpain, 2001; Vilei et al., 1997). Tra-3, a non-EF-hand calpain since it lacks a calmodulin-like region (domain IV) (Huang and involved in sex determination in C. elegans, is activated by Wang, 2001). Other members of this group are mammalian Ca2+ (Sokol and Kuwabara, 2000). These data suggest that Ha- calpains 5, 6, 7, 10 and 13 (SOLH), Caenorhabditis elegans CalpM has Ca2+-dependent proteolytic activity, although this tra-3, and Drosophila Sol (Sorimachi and Suzuki, 2001). must be demonstrated on expressed recombinant protein using Isoforms of Drosophila CalpA (Dm-CalpA′; estimated mass functional assays. 64 kDa) and mammalian CAPN2 and CAPN3 lacking domain The Ha-CalpM cDNA appears to encode CDP III, one of IV result from alternative mRNA splicing, which introduces a four calpain activities identified in lobster skeletal muscles termination codon near the C-terminal end of domain III (Mykles and Skinner, 1986). The Ha-CalpM isoforms elute Muscle-specific calpain expression in Homarus americanus 571

Fig. 10. Gel filtration chromatography of Ha-CalpM. A calpain fraction from Q Sepharose ion exchange chromatography was separated on a Superdex-200 gel filtration column (see Materials and methods). 10 µl samples of odd-numbered fractions were separated on 10% SDS- polyacrylamide gels and either stained with silver (A) or blotted onto PVDF membrane and probed with Ha-CalpM antiserum (B). The antibody recognized two Ha-CalpM isoforms of 62 kDa and 68 kDa (B, arrows) that co-eluted from the column. The smaller molecular mass immunoreactive polypeptides are probably degradation products. The arrow in A indicates the position of the 62 kDa isoform in the silver- stained gel. The elution position of 62 kDa and 68 kDa isoforms (maximum at fraction 21) corresponded to that of CDP III (estimated mass 59 kDa) (Mykles, 2000; Mykles and Skinner, 1986). The peak CDP IIa activity (125 kDa) elutes at fraction 15 and the peak CDP IIb activity (195 kDa) elutes at fraction 12 (Mykles, 2000). Positions of molecular mass standards (kDa) are indicated. from a gel filtration column at the same position as CDP III elute through several chromatographic separations. The (59 kDa). The other three calpains (CDP I, 310 kDa; CDP IIa, expression of the two isoforms is not correlated with the fiber 125 kDa; and CDP IIb, 195 kDa) elute earlier from the column type composition of the muscles. The 62 kDa isoform occurs (Mykles, 2000). The elution position of the Ha-CalpM proteins in both crusher claw closer muscle, which contains only slow from the Mono Q column differed from those of CDPs IIa and fibers, and cutter claw closer muscle, which contains mostly IIb; under identical conditions, CDP IIa elutes at fractions fast fibers (Mykles, 1985). The 62 kDa isoform could either be 17–18 and CDP IIb at fractions 27–28 (Beyette et al., 1997; the active form of calpain produced by autolysis of the 68 kDa Mykles, 2000). Moreover, the 68 kDa isoform is similar to the isoform or an isoform generated by alternative splicing or post- predicted mass (66.3 kDa) of the deduced amino acid sequence translational processing. of Ha-CalpM. These data indicate that the native CDP III A three-dimensional model of Ha-CalpM, using the crystal comprises a single polypeptide encoded by the Ha-CalpM structure of mammalian m-calpain (Capn2) as template, is gene. CDP IIb is a homodimer of a 95 kDa subunit, while CDP presented in Fig. 12. The colors denote aa residues that are IIa is a homodimer of a 60 kDa subunit; both calpains appear identical between Ha-CalpM and human m-calpain. The first to be products of distinct genes (Beyette et al., 1997). The 75-aa acid sequence of domain I is unique, showing no subunit composition of CDP I is not known. However, the sequence identity with any other gene in the NCBI database. identity of Ha-CalpM and CDP III remains to be established. Consequently, the first 60 residues could not be We were unable to obtain N-terminal sequences of the 62 kDa accommodated in the model. The last 35-aa sequence of and 68 kDa isoforms by Edman degradation, suggesting that domain I has 40% and 49% identity with the same region of the N termini were chemically blocked. Drosophila calpains A and B, respectively. Domain II is The 62 kDa and 68 kDa isoforms are probably encoded by divided into two subdomains (IIa and IIb), which form the the same gene, since both proteins react specifically to active site (Fig. 12A) (Hosfield et al., 1999; Strobl et al., antibody raised against the unique N-terminal sequence and co- 2000). The Cys141 in domain IIa and the His299 and Asn329 572 X. Yu and D. L. Mykles

Fig. 11. Ion-exchange chromatography of Ha-CalpM. Fractions 17–23 from gel filtration chromatography (Fig. 10) were pooled and separated on a Mono Q column (see Materials and methods). 10 µl samples of odd-numbered fractions between fractions 7 and 35 were separated on 10% SDS-polyacrylamide gels and either stained with silver (A) or blotted onto PVDF membrane and probed with Ha-CalpM antiserum (B). The arrows indicate the positions of the 62 kDa and 68 kDa Ha-CalpM isoforms. The elution position of Ha-CalpM (fractions 23–25; 0.35–0.37 mol l–1 NaCl) differed from that of CDP IIb (fractions 27–28; 0.40–0.41 mol l–1 NaCl) and CDP IIa (fractions 17–18; 0.28 mol l–1 NaCl) under identical conditions (Beyette et al., 1997; Mykles, 2000). Positions of molecular mass standards (kDa) are indicated. in domain IIb form a catalytic triad essential for cysteine movement of the Asn329 and His299 on one side of the proteinase activity (Arthur et al., 1995). Many of the catalytic cleft toward the Cys141 on the other, thus forming conserved residues are located in the vicinity of the catalytic of a functional catalytic site (Fig. 12A). site (Fig. 12A). The most striking feature of Ha-CalpM exists Calpain genes show a range of tissue expression, from in domain III, which contains a highly acidic sequence ubiquitous to highly tissue-specific. The mammalian calpains consisting of a stretch of 17 aspartate residues (Fig. 12B). All 1 (µ-calpain), 2 (m-calpain), 5, 7, 10, 12 and 13, are calpains have an acidic loop as part of the C2-like domain, but ubiquitously expressed, although calpain 5 is highly expressed the expansion of this acidic stretch is unique to Ha-CalpM. A in colon, small intestine and testis and calpain 12 is highly repeat sequence of 44 aspartate residues has been reported in expressed in hair follicle (Huang and Wang, 2001; Sorimachi calsequestrin, a high-capacity, moderate-affinity, Ca2+- and Suzuki, 2001). The preferential expression of Ha-CalpM binding protein that acts as an internal Ca2+ store in fast in skeletal muscle is not unusual. Alternatively spliced muscle fibers (Treves et al., 1992). Analysis of the crystal isoforms of mammalian CAPN3 are restricted to skeletal structure of m-calpain suggests that the acidic region interacts muscle (p94 calpain), lens (Lp82 and Lp85 calpains) and retina with Ca2+ and functions as an ‘electrostatic switch’ for (Rt88 calpain) (Huang and Wang, 2001; Sorimachi and Suzuki, regulating proteinase activity (Hosfield et al., 2001; Strobl et 2001). Mammalian calpain 6 is expressed in placenta, calpain al., 2000). The X-ray structures of rat and human m-calpain 8 in stomach mucosa, calpain 9 in digestive tract, and calpain also show that domain III is engaged in extensive ionic 11 in intestine (Dear and Boehm, 1999; Lee et al., 1999). In interactions with all the other domains within the large subunit Drosophila, Dm-CalpA is differentially expressed during and thus seems to function as an organizing center of calpain development (Emori and Saigo, 1994; Theopold et al., 1995). (Hosfield et al., 1999; Strobl et al., 2000). These data suggest Although the Dm-CalpA mRNA is ubiquitously expressed in that domain III of Ha-CalpM plays a significant role for full adult flies, immunocytochemistry shows the protein is activation of the enzyme. Ca2+ binding would result in restricted to cells in the brain, ventral ganglion and blood Muscle-specific calpain expression in Homarus americanus 573

Fig. 12. Model of Ha-CalpM, based on the crystal structure of mammalian m-calpain (CAPN2) in the absence of Ca2+. Identical residues between the lobster and human deduced amino acid sequences are colored (red, acidic residues; blue, basic; yellow, hydrophobic; lavender, uncharged polar). (A) ‘Top’ view of the catalytic domain (domain II) containing the three conserved residues (C141, H299 and N329, in green) forming the catalytic triad of the active site. The residues of the triad are surrounded by conserved nonpolar residues, which form a hydrophobic environment within the catalytic cleft. (B) ‘Side’ view showing an acidic loop (magenta and red) containing 19 acidic residues in a stretch of 20 residues (D452, D455, D457 and D458 are not colored because they did not align with identical residues in the human m- calpain; see Fig. 2). The acidic loop, which includes a stretch of 11 aspartate residues (magenta; residues 460–470) unique to the lobster sequence, is part of a putative calcium-dependent phospholipid-binding C2-like region in domain III. Binding of Ca2+ to the acidic loop and to two aspartates (Fig. 2; D232 and D366) in domain II results in closure of the catalytic cleft to allow cooperation between C141, H299 and N329 necessary for catalytic activity (Hosﬁeld et al., 2001; Moldoveanu et al., 2002). The ﬁrst 60 residues of the N-terminal sequence were excluded, due to its high divergence from the mammalian sequence (Fig. 2). The lobster sequence lacks a calmodulin-like domain (domain IV).

(hemocytes) (Theopold et al., 1995). These data suggest that CalpM mRNA and protein levels were not elevated in premoult certain calpains mediate tissue-specific functions. muscle, the muscle-specific expression of Ha-CalpM (Table 1; The size (about 2 kb) of the Ha-CalpM cDNA is smaller than Figs 4–6), and the ability of Ha-CalpM/CDP III to degrade the transcript sizes in northern blots. Two major transcripts of myofibrillar proteins (Mattson and Mykles, 1993), suggests Ha-CalpM (6.1 kb and 3.3 kb) were detected at high stringency. that it has a role in remodeling and/or maintaining the structure An additional minor transcript (4 kb) was expressed in fast of the contractile apparatus. This is analogous to the p94 muscle. This suggests that the three transcripts contain the Ha- isoform of CAPN3 in mammalian skeletal muscle. The activity CalpM ORF but differ in the lengths of the UTRs. The 3′ UTR of p94 is necessary to maintain muscle fiber viability and of the Ha-CalpM cDNA lacks a polyadenylation signal and a myofibrillar protein composition. Loss-of-function mutations poly(A) tail. Given the high percentage (40%) of adenosines in the CAPN3 gene are the underlying cause of limb-girdle in the 3′ UTR, the oligo(dT) primer used for the initial cDNA muscular dystrophy type 2A in humans (Chae et al., 2001). synthesis may have annealed to this region, rather than to a Expression of CAPN3 responds rapidly to conditions that poly(A) tail located further downstream. This suggests that induce muscle atrophy or degeneration. Denervation or muscle the 3′ UTR of the Ha-CalpM mRNA is longer than 234 bp. injury result in a rapid decrease in CAPN3 mRNA in mouse The three transcript sizes may arise from alternative skeletal muscle (Stockholm et al., 2001), whereas sepsis causes polyadenylation signals in the 3′ UTR. an increase in CAPN3 mRNA in rat skeletal muscle (Williams Crustacean muscles contain four calpain activities that are et al., 1999). The p94 appears to maintain the structural involved in the degradation of myofibrillar proteins during integrity of the contractile apparatus through its interaction programmed claw muscle atrophy (Mykles, 1999). Coincident with connectin/titin (Herasse et al., 1999). with this degradation is a substantial restructuring of the In summary, the full-length cDNA encoding a non-EF-hand myofibrils. Myofibrillar cross-sectional area decreases 78%, calpain expressed in lobster skeletal muscle has been the thin:thick myofilament ratio decreases from 9:1 to 6:1, and characterized. This is the first calpain gene isolated from a thick myofilament packing increases by 51% to 72% (Ismail crustacean species. It appears to encode the CDP III activity and Mykles, 1992; Mykles and Skinner, 1981). Although Ha- characterized in biochemical studies. The Ha-CalpM is 574 X. Yu and D. L. Mykles expressed as a 62 kDa isoform in claw muscles and a 68 kDa through tissue-specific transcriptional and posttranscriptional events. Mol. isoform in deep abdominal muscle; the functional significance Cell. Biol. 19, 4047-4055. Hertz-Fowler, C., Ersfeld, K. and Gull, K. (2001). CAP5.5, a life-cycle- of the two isoforms is not known. Although the four crustacean regulated, cytoskeleton-associated protein is a member of a novel family of calpains appear to be products of different genes, the precise calpain-related proteins in Trypanosoma brucei. Mol. Biochem. Parasitol. relationship between Ha-CalpM and CDPs I, IIa and IIb will 116, 25-34. Hosfield, C. M., Elce, J. S., Davies, P. L. and Jia, Z. C. (1999). Crystal not be established until the full-length sequences of the structure of calpain reveals the structural basis for Ca2+-dependent protease remaining calpains are determined. Our data suggest that Ha- activity and a novel mode of enzyme activation. EMBO J. 18, 6880-6889. CalpM plays a role in restructuring the skeletal muscle during Hosfield, C. M., Moldoveanu, T., Davies, P. L., Elce, J. S. and Jia, Z. C. (2001). Calpain mutants with increased Ca2+ sensitivity and implications for the moulting cycle. However, its specific role will remain the role of the C2-like domain. J. Biol. Chem. 276, 7404-7407. undefined until endogenous substrates are identified. Huang, Y. H. and Wang, K. K. W. (2001). The calpain family and human disease. Trends Mol. Med. 7, 355-362. Ismail, S. Z. M. and Mykles, D. L. (1992). Differential molt-induced atrophy We thank Dr Ernest S. Chang, Bodega Marine Laboratory, in the dimorphic claws of male fiddler crabs, Uca pugnax. J. Exp. Zool. 263, University of California, Davis, for providing lobsters at 18-31. different moulting stages; Dr Scott Medler and Ms Kitty Jékely, G. and Friedrich, P. (1999). Characterization of two recombinant Drosophila calpains – CALPA and a novel homolog, CALPB. J. Biol. Brown for technical assistance; and Drs John Elce and Chem. 274, 23893-23900. Zongchao Jia, Queen’s University, and Dr Karolin Luger, Kabsch, W. and Sander, C. (1983). Dictionary of protein secondary Colorado State University, for their help in modeling the structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577-2637. lobster Ha-CalpM. This research was supported by a grant Khorchid, A. and Ikura, M. (2002). How calpain is activated by calcium. from National Science Foundation (IBN-9904528). Nature Struct. Biol. 9, 239-241. Koenders, A., Yu, X. L., Chang, E. S. and Mykles, D. L. (2002). Ubiquitin and actin expression in claw muscles of land crab, Gecarcinus lateralis, and American lobster, Homarus americanus: differential expression of ubiquitin References in two slow muscle fibre types during moult-induced atrophy. J. Exp. Zool. Andresen, K., Tom, T. D. and Strand, M. (1991). Characterization of cDNA 292, 618-632. clones encoding a novel calcium-activated neutral proteinase from Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly Schistosoma mansoni. J. Biol. Chem. 266, 15085-15090. of the head of bacteriophage T4. Nature 227, 680-685. Arthur, J. S. C., Gauthier, S. and Elce, J. S. (1995). Active site residues in Lee, H. J., Tomioka, S., Kinbara, K., Masumoto, H., Jeong, S. Y., m-calpain: Identification by site-directed mutagenesis. FEBS Lett. 368, 397- Sorimachi, H., Ishiura, S. and Suzuki, K. (1999). Characterization of a 400. human digestive tract-specific calpain, nCL-4, expressed in the baculovirus Avruch, J. and Wallach, D. F. H. (1971). Preparation and properties of system. Arch. Biochem. Biophys. 362, 22-31. plasma membrane and endoplasmic reticulum fragments from isolated rat Mattson, J. M. and Mykles, D. L. (1993). Differential degradation of fat cells. Biochim. Biophys. Acta 233, 334-337. myofibrillar proteins by four calcium-dependent proteinase activities from Beyette, J. R., Emori, Y. and Mykles, D. L. (1997). Immunological analysis lobster muscle. J. Exp. Zool. 265, 97-106. of two calpain-like Ca2+-dependent proteinases from lobster striated Moldoveanu, T., Hosfield, C. M., Lim, D., Elce, J. S., Jia, Z. C. and Davies, muscles: Relationship to mammalian and Drosophila calpains. Arch. P. L. (2002). A Ca2+ switch aligns the active site of calpain. Cell 108, 649- Biochem. Biophys. 337, 232-238. 660. Beyette, J. R., Ma, J.-S. and Mykles, D. L. (1993). Purification and autolytic Mykles, D. L. (1985). Multiple variants of myofibrillar proteins in single fibers degradation of a calpain-like calcium-dependent proteinase from lobster of lobster claw muscles: evidence for two types of slow fibers in the cutter (Homarus americanus) striated muscle. Comp. Biochem. Physiol. 104B, 95- closer muscle. Biol. Bull. 169, 476-483. 99. Mykles, D. L. (1988). Histochemical and biochemical characterization of Beyette, J. R. and Mykles, D. L. (1997). Autolysis and biochemical two slow fiber types in decapod crustacean muscles. J. Exp. Zool. 245, properties of a lobster muscle calpain-like proteinase. J. Exp. Zool. 277, 232-243. 106-119. Mykles, D. L. (1990). Calcium-dependent proteolysis in crustacean claw Beyette, J. R. and Mykles, D. L. (1999). Crustacean calcium-dependent closer muscle maintained in vitro. J. Exp. Zool. 256, 16-30. proteinases. In The Pharmacology of Calpain (ed. K. K. W. Wang and P.- Mykles, D. L. (1998). Intracellular proteinases of invertebrates: calcium- W. Yuen), pp. 409-427. Washington, DC: Taylor & Francis. dependent and proteasome/ubiquitin-dependent systems. Int. Rev. Cytol. Chae, J., Minami, N., Jin, Y., Nakagawa, M., Murayama, K., Igarashi, F. 184, 157-289. and Nonaka, I. (2001). Calpain 3 gene mutations: genetic and clinico- Mykles, D. L. (1999). Proteolytic processes underlying molt-induced claw pathologic findings in limb-girdle muscular dystrophy. Neuromusc. Disord. muscle atrophy in decapod crustaceans. Am. Zool. 39, 541-551. 11, 547-555. Mykles, D. L. (2000). Purification and characterization of crustacean calpain- Cotton, J. L. S. and Mykles, D. L. (1993). Cloning of a crustacean myosin like proteinases. Meth. Mol. Biol. 144, 55-66. heavy chain isoform: exclusive expression in fast muscle. J. Exp. Zool. 267, Mykles, D. L., Haire, M. F. and Skinner, D. M. (2000). 578-586. Immunocytochemical localization of actin and tubulin in the integument of Dear, T. N. and Boehm, T. (1999). Diverse mRNA expression patterns of the land crab (Gecarcinus lateralis) and lobster (Homarus americanus). J. Exp. mouse calpain genes Capn5, Capn6 and Capn11 during development. Mech. Zool. 286, 329-342. Dev. 89, 201-209. Mykles, D. L. and Skinner, D. M. (1981). Preferential loss of thin filaments Dutt, P., Arthur, J. S. C., Grochulski, P., Cygler, M. and Elce, J. S. (2000). during molt-induced atrophy in crab claw muscle. J. Ultrastr. Res. 75, 314- Roles of individual EF-hands in the activation of m-calpain by calcium. 325. Biochem. J. 348, 37-43. Mykles, D. L. and Skinner, D. M. (1982). Molt cycle-associated changes in Emori, Y. and Saigo, K. (1994). Calpain localization changes in coordination calcium-dependent proteinase activity that degrades actin and myosin in with actin-related cytoskeletal changes during early embryonic development crustacean muscle. Dev. Biol. 92, 386-397. of Drosophila. J. Biol. Chem. 269, 25137-25142. Mykles, D. L. and Skinner, D. M. (1983). Ca2+-dependent proteolytic activity Hata, S., Sorimachi, H., Nakagawa, K., Maeda, T., Abe, K. and Suzuki, in crab claw muscle. Effects of inhibitors and specificity for myofibrillar K. (2001). Domain II of m-calpain is a Ca2+-dependent cysteine protease. proteins. J. Biol. Chem. 258, 10474-10480. FEBS Lett. 501, 111-114. Mykles, D. L. and Skinner, D. M. (1986). Four Ca2+-dependent proteinase Herasse, M., Ono, Y., Fougerousse, F., Kimura, E., Stockholm, D., Beley, activities isolated from crustacean muscle differ in size, net charge, and C., Montarras, D., Pinset, C., Sorimachi, H., Suzuki, K. et al. (1999). sensitivity to Ca2+ and inhibitors. J. Biol. Chem. 261, 9865-9871. Expression and functional characteristics of calpain 3 isoforms generated Shean, B. S. and Mykles, D. L. (1995). Polyubiquitin in crustacean striated Muscle-specific calpain expression in Homarus americanus 575

muscle: Increased expression and conjugation during molt-induced claw an electrostatic switch mechanism for activation by calcium. Proc. Natl. muscle atrophy. Biochim. Biophys. Acta 1264, 312-322. Acad. Sci. USA 97, 588-592. Skinner, D. M. (1966). Breakdown and reformation of somatic muscle during Theopold, U., Pintér, M., Daffre, S., Tryselius, Y., Friedrich, P., Nässel, the molt cycle of the land crab, Gecarcinus lateralis. J. Exp. Zool. 163, 115- D. R. and Hultmark, D. (1995). CalpA, a Drosophila calpain homolog 124. specifically expressed in a small set of nerve, midgut, and blood cells. Mol. Sokol, S. B. and Kuwabara, P. E. (2000). Proteolysis in Caenorhabditis Cell. Biol. 15, 824-834. elegans sex determination: cleavage of TRA-2A by TRA-3. Genes Dev. 14, Towbin, H., Stahelin, T. and Gordon, J. (1979). Electrophoretic transfer of 901-906. protein from polyacrylamide gels to nitrocellulose sheets: procedure and Sorimachi, H., Ishiura, S. and Suzuki, K. (1993). A novel tissue-specific some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. calpain species expressed predominantly in the stomach comprises two Treves, S., Vilsen, B., Chiozzi, P., Andersen, J. P. and Zorzato, F. (1992). alternative splicing products with and without Ca2+-binding domain. J. Biol. Molecular cloning, functional expression and tissue distribution of the Chem. 268, 19476-19482. cDNA encoding frog skeletal muscle calsequestrin. Biochem. J. 283, 767- Sorimachi, H. and Suzuki, K. (2001). The structure of calpain. J. Biochem. 772. (Tokyo) 129, 653-664. Vilei, E. M., Calderara, S., Anagli, J., Berardi, S., Hitomi, K., Maki, M. Stockholm, D., Herasse, M., Marchand, S., Praud, C., Roudaut, C., and Carafoli, E. (1997). Functional properties of recombinant calpain I and Richard, I., Sebille, A. and Beckmann, J. S. (2001). Calpain 3 mRNA of mutants lacking domains III and IV of the catalytic subunit. J. Biol. Chem. expression in mice after denervation and during muscle regeneration. Am. 272, 25802-25808. J. Physiol. 280, C1561-C1569. Williams, A. B., Decourten-Myers, G. M., Fischer, J. E., Luo, G. J., Sun, Strobl, S., Fernandez-Catalan, C., Braun, M., Huber, R., Masumoto, H., X. Y. and Hasselgren, P. O. (1999). Sepsis stimulates release of Nakagawa, K., Irie, A., Sorimachi, H., Bourenkow, G., Bartunik, H. et myofilaments in skeletal muscle by a calcium-dependent mechanism. al. (2000). The crystal structure of calcium-free human m-calpain suggests FASEB J. 13, 1435-1443.